title: Giardia duodenalis typing from stools: a comparison of three approaches to extracting DNA, and validation of a probe-based real-time PCR typing assay.
abstract: A weighted, multi-attribute approach was used to compare three methods for direct extraction of Giardia duodenalis DNA from 15 microscopy-positive stools: (1) a QIAamp spin-column method for stools including a 10 min incubation at 95 °C, (2) method 1 preceded by five freeze-thaw cycles and (3) bead beating with guanidine thiocyanate using a FastPrep-28 machine followed by liquid-phase silica purification of DNA. The attributes compared included DNA yield measured using a new triose phosphate isomerase (tpi) gene probe-based real-time PCR, also described here. All three methods shared 100 % PCR positivity, while the bead-beating method provided the highest G. duodenalis DNA yield (P<0.01). However, when other weighted attributes, including biocontainment, resources and technical requirements, were also considered, spin-column extraction with prior freeze-thaw treatment (method 2) was deemed the most desirable and was selected for use. The tpi real-time PCR typing assay was designed to discriminate between the main human infectious assemblages of G. duodenalis (A and B) and was evaluated initially using standard isolates. Validation using microscopy-positive stools from 78 clinical giardiasis cases revealed 100 % typability; 20 (26 %) samples contained assemblage A, 56 (72 %) assemblage B and two (3 %) assemblages A and B. While the epidemiological significance of assemblage distribution will be revealed as more isolates are typed and analysed with patient demographic and exposure data, the utility of this assay and its ready application in our laboratory workflow and result turnaround margins is already evident.
authors.affiliation: Cryptosporidium Reference Unit, Public Health Wales Microbiology, Singleton Hospital, Swansea SA2 8QA, UK.
authors.forename: K
authors.initials: K
authors.lastname: Elwin
authors.affiliation: Division of Infection & Immunity, Research Department of Infection, University College London, London WC1E 6BT, UK.
authors.forename: H V
authors.initials: HV
authors.lastname: Fairclough
authors.affiliation: Cryptosporidium Reference Unit, Public Health Wales Microbiology, Singleton Hospital, Swansea SA2 8QA, UK.
authors.forename: S J
authors.initials: SJ
authors.lastname: Hadfield
authors.affiliation: Cryptosporidium Reference Unit, Public Health Wales Microbiology, Singleton Hospital, Swansea SA2 8QA, UK.
authors.forename: R M
authors.initials: RM
authors.lastname: Chalmers
mesh_terms: D000293:Adolescent; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D002648:Child; D002675:Child, Preschool; D016054:DNA, Protozoan / Q000235:genetics / Q000302:isolation & purification*; D005243:Feces / Q000469:parasitology*; D005260:Female; D016829:Giardia lamblia / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification; D005873:Giardiasis / Q000469:parasitology*; D006801:Humans; D007223:Infant; D008297:Male; D008875:Middle Aged; D017720:Molecular Epidemiology / Q000379:methods; D010274:Parasitology / Q000379:methods*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D013048:Specimen Handling / Q000379:methods*; D055815:Young Adult
chemical_list: D016054:DNA, Protozoan